Studying Bacterial Pathogenesis: Approaches and Methods Flashcards
Why is it important to study bacterial pathogenesis?
To improve prevention, diagnosis and treatment of bacterial disease
How can bacteria disease be prevented?
Vaccines
How can bacterial disease be diagnosed?
Toxin detection
How can bacterial disease be treated?
Identifying new therapy targets
What is bacterial pathogenicity?
The ability of a bacterium to inflict damage on the host
What does bacterial pathogenesis involve?
Bacterial and host factors
Give examples of bacterial factors
Virulence factors
Toxins
Immune evasion
Attachment
Motility
Gene regulation
Spread
Resistance
Give examples of host factors
Immune response
Skin
Phagocytes
Complement
Fe restriction
Abs
Lymphocytes
Macrophages
How can we prove that a bacterium is responsible for a particular disease?
Using Koch’s postulates:
1. Bacterium is found in all people with the disease
2. The bacterium can be isolated from patients and maintained in pure culture
3. The pure culture can be inoculated into a human volunteer or animal model and cause symptoms of the disease
4. The bacterium can be re-isolated from the volunteer or animal
e.g. Helicobacter pylori
What is needed to investigate bacterial pathogenesis?
Clinical observation and epidemiology
Models of disease - in vivo and in vitro
Appropriate strains of bacteria to test - inc geneticall modified bacteria
What is epidemiology?
Study of spread and distribution of disease
In bacteriology it often includes the discrimination of different strains/variants and their spread
How is clinical observation and epidemiology done?
Identifying the problem
Identifying common features of cases
Determining which tissues are targeted
Identifying if all people are equally susceptible
Identifying how it spreads
Sources of outbreaks
Causative organism
Identifying if all strains are equally pathogenic
How are good epidemiological studies designed?
Clear definitions of patients with and without the disease
Sufficient numbers of patient to investigate - statistical significance
Collection of relevant information and samples
Consider ethics, patients are complex, logistics
What are the different models of disease?
Bacterial behaviour in rich or specialised growth media (in vitro)
Bacterial behaviour in laboratory conditions (in vitro) that mimic in vivo
Animal models (in vivo)
Describe behaviour on/in rich or specialised growth media
e.g. agar plates or tryptone soy broth medium (TSB)
What agar plates/specialised growth media useful to identify?
Growth requirements - nutrients, O2, CO2, Fe
Motility
Ability to digest proteins, lipids, carbs, DNA, etc.
Adherence
Morphology - e.g. flagella, capsule
What are advantages of observing behaviour on rich or specialised growth media?
Inexpensive
Large scale is possible
Assays are flexible
Reagents easily available
What are disadvantages of observing behaviour on rich/specialised growth media?
Does not mimic in vivo situation:
- no host interaction
- bacterial gene regulation is dependent on external conditions, but rich conditions are not representative of infection
How does bacterial gene regulation work?
Two component signal transduction
What are examples of growing bacteria in lab conditions that mimic in vivo?
Blood, plasma, serum
Peritoneal dialysis fluid
Fresh mammalian cells
Cell culture
Tissue culture
What are advantages of growing bacteria in lab conditions that mimic in vivo?
Mimic host interaction and growth conditions
What are disadvantages of growing bacteria in lab conditions that mimic in vivo?
More expensive
Limited access to reagents
Hard to scale-up
Not as reproduceable
Examples of animal models
Mice
Rats
Rabbits
Larger animals
Flies
Worms
What is the method for using animal models?
Purified bacteria inoculated into the animal - can be intraperitoneally, intravenously, orally etc. (may depend on disease)
Inoculum size/number of bacteria is important
How is the outcome measured for animal models?
ID50
LD50
Symptoms
Cfu in tissue
Migration of immune cells
Ab response
Cytokine response
Advantages of animal models
Best available mimic of human infection in vivo
Transgenic animals available
Essential for trials of therapies, safety - required before human studies
Disadvantages of animal models
Ethics - licensing, restricted numbers, animals must not suffer unnecessarily
How should bacteria be inoculated
What outcomes need to be measured
Variation between different models
What are the different bacterial genetics methods and their uses?
Sequencing projects - what is the bacteria capable of
Comparative genomics - are all the strains the same
Cloning - can addition of 1 gene make a bacterium pathogenic
Isogenic mutants (knockouts) - can deletion of 1 gene make a bacterium non-pathogenic
Transposon library screening - which genes in the genome are responsible for a phenotype/virulence
Transcriptional genomics - which genes are expressed during infection
